SUMMARY The gastrointestinal (GI) tract is a major site of Human Immunodeficiency Virus (HIV) replication, which leads to intestinal inflammation, CD4+ T cell depletion and gut epithelial barrier dysfunction. These defects are thought to drive HIV disease progression by allowing translocation of luminal microbial products into the circulation, which triggers chronic systemic immune activation and disease progression. Although antiretroviral therapy (ART) effectively suppress viral replication in the blood, it does not restore CD4+ T cell levels in the gut; even individuals with suppressed viral loads for decades have persistent gut barrier impairment and continued microbial translocation. Epithelial intestinal stem cells (ISCs) are responsible for replenishing the intestinal epithelium during normal homeostatic turnover and orchestrating epithelial repair following injury. Current studies of intestinal epithelial dysfunction in HIV are limited and have relied primarily on correlative observations based on histology or peripheral biomarkers. Our proposal addresses critical gaps in knowledge by 1) performing in-depth characterization of ISC proliferation, self-renewal and differentiation ex vivo and in vivo in HIV-infected and uninfected individuals; 2) utilizing tissue samples from a unique cohort of individuals in sub-Saharan Africa, where HIV burden is highest and most devastating; 3) applying cutting-edge techniques to uncover molecular signalling pathways involved in HIV-induced epithelial dysfunction; and 4) thoroughly testing mechanistic hypotheses using relevant mouse models and ex vivo organoid culture systems. With our combined expertise in HIV disease, mucosal immunology and GI pathology, we are well positioned to uncover specific mechanisms that underlie intestinal epithelial dysfunction in HIV infection. An understanding of these mechanisms is critical to facilitate development of novel therapeutic approaches to improve health outcomes of people living with HIV. In this proposal, we hypothesize that HIV infection results in hyperproliferation of ISCs, aberrant ISC differentiation, increase epithelial death, and dysfunction of epithelial cellular junction integrity. Additionally, based upon our data, we hypothesize that this HIV-associated ISC dysfunction can occur via two mechanisms: 1) depletion of intestinal CD4+ T cells and loss of specific T helper cytokines that promote intestinal epithelial homeostasis and 2) type I and II interferon signalling within ISCs in HIV-infected individuals. To address these hypotheses we are proposing two complementary aims. Aim 1 will use state-of-the-art technologies to characterise ISC function directly ex vivo in gut pinch biopsies sampled throughout the intestinal mucosa from our established cohort at Albert Luthuli Central Hospital in Durban, South Africa. In Aim 2 will use in vivo mouse models of CD4+ T cell depletion and examine the impact on ISC proliferation, self-renewal and differentiation, and use ex vivo human organoid models to study the impact of type I and II interferon pathways on ISC function. This proposal will address important unknown mechanisms of ISC biology that may help in the development of new strategies to reverse gut barrier defects in HIV infection.